Ever since it was invented in the early 1990s (Science, 251, 767-773, 1991), high-density arrays formed by spatially addressable synthesis of bioactive probes on a 2-dimensional solid support has greatly enhanced and simplified the process of biological research and development. The key to current microarray technology is deposition of a bioactive agent at a single spot on a microchip in a “spatially addressable” manner.
Current technologies have used various approaches to fabricate microarrays. For example, U.S. Pat. Nos. 5,412,087, and 5,489,678 demonstrate the use of a photolithographic process for making peptide and DNA microarrays. The patent teaches the use of photolabile protecting groups to prepare peptide and DNA microarrays through successive cycles of deprotecting a defined spot on a 1 cm×1 cm chip by photolithography, then flooding the entire surface with an activated amino acid or DNA base. Repetition of this process allows construction of a peptide or DNA microarray with thousands of arbitrarily different peptides or oligonucleotide sequences at different spots on the array. This method is expensive. An ink jet approach is being used by others (e.g., U.S. Pat. Nos. 6,079,283; 6,083,762; and 6,094,966) to fabricate spatially addressable arrays, but this technique also suffers from high manufacturing cost in addition to the relatively large spot size of 40 to 100 μm. Because the number of bioactive probes to be placed on a single chip usually runs anywhere from 1000 to 100000 probes, the spatial addressing method is intrinsically expensive regardless how the chip is manufactured. An alternative approach to the spatially addressable method is the concept of using fluorescent dye-incorporated polymeric beads to produce biological multiplexed arrays. U.S. Pat. No. 5,981,180 discloses a method of using color coded beads in conjunction with flow cytometry to perform multiplexed biological assay. Micro-spheres conjugated with DNA or monoclonal antibody probes on their surfaces were dyed internally with various ratios of two distinct fluorescence dyes. Hundreds of “spectrally addressed” micro-spheres were allowed to react with a biological sample and the “liquid array” was analyzed by passing a single micro-sphere through a flow cytometry cell to decode sample information. U.S. Pat. No. 6,023,540 and 6,266,459 discloses the use of fiber-optic bundles with pre-etched microwells at distal ends to assemble dye loaded micro-spheres. The surface of each spectrally addressed micro-sphere was attached with a unique bioactive agent and thousands of micro-spheres carrying different bioactive probes combined to form “beads array” on pre-etched microwells of fiber optical bundles. More recently, a novel optically encoded micro-sphere approach was accomplished by using different sized zinc sulfide-capped cadmium selenide nanocrystals incorporated into micro-spheres (Nature Biotech. 19, 631-635, 2001). Given the narrow band width demonstrated by these nanocrystals, this approach significantly expands the spectral bar coding capacity in micro-spheres.
Even though the “spectrally addressed micro-sphere” approach does provide an advantage in terms of its simplicity over the old fashioned “spatially addressable” approach in microarray making, there was still needs in the art to make the manufacture of biological microarrays less difficult and less expensive and to provide nucleic acid identification systems that are accurate, less complex and less expensive.
U.S. Ser. No. 09/942,241 provides a microarray that is less costly and easier to prepare than those previously disclosed because the support need not be modified; nevertheless the micro-spheres remain immobilized on the substrate.
U.S. Ser. No. 09/942, 241 provides a microarray comprising: a substrate coated with a composition comprising micro-spheres dispersed in a fluid containing a gelling agent or a precursor to a gelling agent, wherein the micro-spheres are immobilized at random positions on the substrate. The substrate is free of receptors designed to physically or chemically interact with the micro-spheres. That invention utilizes a unique coating composition and technology to prepare a microarray on a substrate that need not be pre-etched with microwells or pre-marked in any way with sites to attract the micro-spheres, as disclosed in the art.
U.S. Ser. No. 09/942,241 teaches various coating methods but exemplifies machine coating, whereby a support is coated with a fluid coating composition comprising micro-spheres dispersed in gelatin. Immediately after coating, the support is passed through a chill set chamber in the coating machine where the gelatin undergoes rapid gelation and the micro-spheres are immobilized.
While that invention provides a huge manufacturing advantage over then existing technologies, it needs some refinement in order to maximize its full potential value to the art. The problem is that during such machine coating and rapid gelation, the gelling agent tends to cover the surface of the micro-spheres, thereby preventing the analyte (such as DNA) from penetrating through the gel overcoat and hybridizing with probes on the surface of the micro-spheres. The gel overcoat problem was solved by using enzyme digestion as disclosed in U.S. Ser. No. 10/062,326. There is a need for a nucleic acid analysis system using such an enzyme treated coated random micro-spheres array in a whole frame imaging capture system.